Dataset Information

HIV-1 Coreceptor Usage Assessment by Ultra-Deep Pyrosequencing and Response to Maraviroc.

ABSTRACT:

Background

Maraviroc is an HIV entry inhibitor that alters the conformation of CCR5 and is poorly efficient in patients infected by viruses that use CXCR4 as an entry coreceptor. The goal of this study was to assess the capacity of ultra-deep pyrosequencing (UDPS) and different data analysis approaches to characterize HIV tropism at baseline and predict the therapeutic outcome on maraviroc treatment.

Methods

113 patients with detectable HIV-1 RNA on HAART were treated with maraviroc. The virological response was assessed at months 1, 3 and 6. The sequence of the HIV V3 loop was determined at baseline and prediction of maraviroc response by different software and interpretation algorithms was analyzed.

Results

UDPS followed by analysis with the Pyrotrop software or geno2pheno algorithm provided better prediction of the response to maraviroc than Sanger sequencing. We also found that the H34Y/S substitution in the V3 loop was the strongest individual predictor of maraviroc response, stronger than substitutions at positions 11 or 25 classically used in interpretation algorithms.

Conclusions

UDPS is a powerful tool that can be used with confidence to predict maraviroc response in HIV-1-infected patients. Improvement of the predictive value of interpretation algorithms is possible and our results suggest that adding the H34S/Y substitution would substantially improve the performance of the 11/25/charge rule.

SUBMITTER: Rodriguez C

PROVIDER: S-EPMC4466260 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Publications

HIV-1 Coreceptor Usage Assessment by Ultra-Deep Pyrosequencing and Response to Maraviroc.

Rodriguez Christophe C Soulié Cathia C Marcelin Anne-Geneviève AG Calvez Vincent V Descamps Diane D Charpentier Charlotte C Flandre Philippe P Recordon-Pinson Patricia P Bellecave Pantxika P Pawlotsky Jean-Michel JM Masquelier Bernard B

PloS one 20150611 6

<h4>Background</h4>Maraviroc is an HIV entry inhibitor that alters the conformation of CCR5 and is poorly efficient in patients infected by viruses that use CXCR4 as an entry coreceptor. The goal of this study was to assess the capacity of ultra-deep pyrosequencing (UDPS) and different data analysis approaches to characterize HIV tropism at baseline and predict the therapeutic outcome on maraviroc treatment.<h4>Methods</h4>113 patients with detectable HIV-1 RNA on HAART were treated with maravir ...[more]

PMID: 26068869

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Project description:INTRODUCTION: Ultra-deep pyrosequencing (UDPS) has been used to detect minority variants within HIV-1 populations. Some aspects of the quality and reproducibility of UDPS have been previously evaluated, but comprehensive studies are still needed. PRINCIPAL FINDING: In this study the UDPS technology (FLX platform) was evaluated by analyzing a 120 base pair fragment of the HIV-1 pol gene from plasma samples from two patients and artificial mixtures of molecular clones. UDPS was performed using an optimized experimental protocol and an in-house data cleaning strategy. Nine samples and mixtures were analyzed and the average number of reads per sample was 19,404 (range 8,858-26,846). The two patient plasma samples were analyzed twice and quantification of viral variants was found to be highly repeatable for variants representing >0.27% of the virus population, whereas some variants representing 0.11-0.27% were detected in only one of the two UDPS runs. Bland-Altman analysis showed that a repeated measurement would have a 95% likelihood to lie approximately within ±0.5 log(10) of the initial estimate. A similar level of agreement was observed for variant frequency estimates in forward vs. reverse sequencing direction, but here the agreement was higher for common variants than for rare variants. UDPS following PCR amplification with alternative primers indicated that some variants may be incorrectly quantified due to primer-related selective amplification. Finally, the in vitro recombination rate during PCR was evaluated using artificial mixtures of clones and was found to be low. The most abundant in vitro recombinant represented 0.25% of all UDPS reads. CONCLUSION: This study demonstrates that this UDPS protocol results in low experimental noise and high repeatability, which is relevant for future research and clinical use of the UDPS technology. The low rate of in vitro recombination suggests that this UDPS system can be used to study genetic variants and mutational linkage.

Project description:BACKGROUND: Ultra-deep pyrosequencing (UDPS) allows identification of rare HIV-1 variants and minority drug resistance mutations, which are not detectable by standard sequencing. PRINCIPAL FINDINGS: Here, UDPS was used to analyze the dynamics of HIV-1 genetic variation in reverse transcriptase (RT) (amino acids 180-220) in six individuals consecutively sampled before, during and after failing 3TC and AZT containing antiretroviral treatment. Optimized UDPS protocols and bioinformatic software were developed to generate, clean and analyze the data. The data cleaning strategy reduced the error rate of UDPS to an average of 0.05%, which is lower than previously reported. Consequently, the cut-off for detection of resistance mutations was very low. A median of 16,016 (range 2,406-35,401) sequence reads were obtained per sample, which allowed detection and quantification of minority resistance mutations at amino acid position 181, 184, 188, 190, 210, 215 and 219 in RT. In four of five pre-treatment samples low levels (0.07-0.09%) of the M184I mutation were observed. Other resistance mutations, except T215A and T215I were below the detection limit. During treatment failure, M184V replaced M184I and dominated the population in combination with T215Y, while wild-type variants were rarely detected. Resistant virus disappeared rapidly after treatment interruption and was undetectable as early as after 3 months. In most patients, drug resistant variants were replaced by wild-type variants identical to those present before treatment, suggesting rebound from latent reservoirs. CONCLUSIONS: With this highly sensitive UDPS protocol preexisting drug resistance was infrequently observed; only M184I, T215A and T215I were detected at very low levels. Similarly, drug resistant variants in plasma quickly decreased to undetectable levels after treatment interruption. The study gives important insights into the dynamics of the HIV-1 quasispecies and is of relevance for future research and clinical use of the UDPS technology.

Dataset Information

HIV-1 Coreceptor Usage Assessment by Ultra-Deep Pyrosequencing and Response to Maraviroc.

Background

Methods

Results

Conclusions

Publications

HIV-1 Coreceptor Usage Assessment by Ultra-Deep Pyrosequencing and Response to Maraviroc.

Similar Datasets

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